Abstract

The steroidogenic factor 1 (SF-1, also known as NR5A1) is a transcription factor belonging to the nuclear receptor superfamily. Whereas most of the members of this family have been extensively characterized, the therapeutic potential and pharmacology of SF-1 still remains elusive. Described here is the identification and characterization of selective inhibitory chemical probes of SF-1 by a rational ultra-high-throughput screening (uHTS) strategy. A set of 64,908 compounds from the National Institute of Health's Molecular Libraries Small Molecule Repository was screened in a transactivation cell-based assay employing a chimeric SF-1 construct. Two analogous isoquinolinones, ethyl 2-[2-[2-(2,3-dihydro-1,4-benzodioxin-7-ylamino)-2-oxoethyl]-1-oxoisoquinolin-5-yl]oxypropanoate (SID7969543) and ethyl 2-[2-[2-(1,3-benzodioxol-5-ylmethylamino)-2-oxoethyl]-1-oxoisoquinolin-5-yl]oxypropanoate and (SID7970631), were identified as potent submicromolar inhibitors, yielding IC(50) values of 760 and 260 nM. The compounds retained their potency in a more physiologic functional assay employing the full-length SF-1 protein and its native response element, yielding IC(50) values of 30 and 16 nM, respectively. The selectivity of these isoquinolinones was confirmed via transactivation-based functional assays for RAR-related orphan receptor A (RORA), Herpes simplex virus transcriptional activator protein Vmw65 (VP16), and liver receptor homolog 1 (LRH-1). Their cytotoxicity, solubility, permeability and metabolic stability were also measured. These isoquinolinones represent valuable chemical probes to investigate the therapeutic potential of SF-1.

Transiently transfected CHO-K1 cells express a chimeric SF-1 nuclear receptor in which the original DNA binding domain (DBD) has been replaced with the Gal4 DBD. Upon interaction with endogenous ligands (L) and/or coactivators (Co-Act) present in CHO-K1 cells, the construct triggers the transcription of a co-transfected luciferase-encoding plasmid via its multimerized Gal4 binding sites. After cell lysis, D-luciferin substrate is used to determine luciferase expression levels by measuring its conversion to light-emitting oxiluciferin. The presence of an SF-1 inhibitor is detected by a decrease of the measured luminescence.

CHO-K1 cells were transiently transfected with the non-fused Gal4 expressing vector pFA-CMV (Vec), or with the Gal4DBD_SF-1LBD expressing plasmid in its wild-type (WT) or mutated (Mut) form. Transfected cells were incubated and assayed for luciferase activity. Luminescent signal is expressed as a percentage of the maximal signal given by WT Gal4DBD_SF-1LBD. Bars represent the mean of three replicates plus or minus their standard deviation. B. Cell seeding density optimization. Both −SF-1 and +SF-1 CHO-K1 cells were seeded in a 1536-well plate at densities ranging from 1,000 to 5,000 cells per well (n=128 wells for each condition). Z’ (circles) and S/B (squares) calculated based on RLUs values measured for −NR and +NR are shown for each tested cell density. The star indicates the selected optimal cell density.

The SF-1 cell-based assay was screened against 64,908 compounds (black dots). The average Z’ value during the screen was 0.72 ± 0.06. The dotted-line represents the activity cutoff, which was calculated at 47.96% inhibition. Compounds with inhibition results above the cutoff are located above the dotted-line.

(A) Comparison of the titration results in the SF-1 and RORA assays. Graphed are the IC50 values of 359 primary hits, titrated in parallel in the SF-1 and RORA assays. Each compound is located according to its IC50 determined in the SF-1 (X-axis) or the RORA (Y-axis) assay. The isoquinolinones SID7969543 and SID7970631 are designated by an arrowhead. (B) Structures of SID7969543 and SID7970631. The isoquinolinone scaffold is indicated in bold.

SID7969543 (A) and SID7970631 (B) were assessed in the SF-1 assay (circles), the RORA assay (squares) and a viability assay (triangles). Additionally (C), SID7969543 (triangles) and SID7970631 (squares) were also assessed in the SFRE/SF-1 assay. Error bars represent the standard error of three separate experiments.